The Indigenous Microbiota - Lab Report Example

Introduction: Human body is a host to a number of bacteria which are referred to as the normal microflora or more specifically the indigenous microbiota of the organ they inhabit, the organs of body normally exposed to outside environment viz. skin, mucous lining, lower GIT, urethra and vagina etc are the parts of body that host these bacteria. Normally the indigenous microbiota share a mutualistic relationship with the host, but some of these bacteria are opportunistic, and cause pathogenesis when the host organ is otherwise weakened. Another cause of pathogenesis can be an infection of pathogenic bacteria which are not normally present in the organ. This could also be the result of loss of normal flora of the organ e.g. Lactobacillus in vagina, which otherwise keep the pathogens population under control. While on one hand it is important to distinguish the normal flora from the pathogens, it is also imperative to identify the pathogen, in order to find the appropriate remedy for the infection. Several microbiological techniques are available for detection, identification and characterization of these microorganisms on the basis of their morphology and specific metabolic processes. Further theses micobes can also be identified on the basis of their specific antibiotic regime. The present experiment deals with deducing the cause of infection of the patients’ Lower GIT and vagina and therefore, samples are taken from the vaginal swabs and faeces of the individual. This would help in determining the cause of the disease and hence providing the appropriate treatment. DISCUSSION: The initial criteria for identification of bacteria isolated from vaginal swab 1 (V1) was it being gram positive. Gram’s staining helps differentiate bacteria on the basis of their cell wall characteristics (Ryan & Ray, 2004). This result excluded all other genera of gram negative bacteria. Cell morphology of V1 being cocci, it is clearly distinguished from gram negative bacilli, can be either of the following: Staphylococcus, Micrococcus, Streptococcus or Enterococcus. The colony morphology: round, raise, smooth, small and cream colour are suggestive of it being either Staphylococci or streptococci. Mannitol salt agar is a selective and differential media for Staphylococci, also differentiating species of Staphylococcus which ferment or do not ferment mannitol. Since V1 is able to grow on Mannitol salt agar and is also able to ferment it, the bacteria V1 can safely be assumed to be Staphylococcus aureus. Since it is catalase positive the option are reduced to two genera, Micrococcus and Staphylococcus, Streptococci are thus excluded.. Oxidase negative result showed that V1 bacteria could be Staphylococcus, thus differentiating it from Micrococcus (Kloos & Bannerman, 1995). Thus the bacteria V1 could be S.aureus. This bacteria is found to be sensitive to the antibiotics ampicillin, gentamycin, tetracycline, cephalothin and chloramphenicol, thus any of these or a combination can be used for the treatment of the infection due to Staphylococcus aureus. However clindamycin cannot be used, since the bacteria was found to be resistant to it. S. aureus is an indigenous microflora of skin mouth and vagina, but causes infection of the skin, UTI etc. The bacteria isolated from Vaginal swab V2 is gram negative, and is also a bacilli, Thus excluding the gram positive and the cocci and restricting search to only gram negative bacilli. V2 give round, raised, smooth, small and creamy colonies, which is suggetive of E. coli. MacConkey agar is a selective and differential media for gram negative bacteria, also differentiating between bacteria that can and cannot ferment lactose, giving a colour change to pink or red due to acid end products of lactose fermentation. V2 is able to grow on MacConkey agar and is also able to ferment lactose, therefore it is confirmed to be a gram negative, lactose fermenting bacteria. A catalse negative test, restricts the result to enterobacteriaceae family. Since the bacteria are non-haemolytic, this added to the fact that it is indole positive we can narrow down the search to Citrobacter diversus, E. coli, Erwinia chrysanthemi and Klebsiella oxytoca; the rest being indole negative. Further biochemical tests (lysine decarboxylase positive, ornithine decarboxylase negative, Glucose conversion to gas, mannitol fermentation and urease negative) confirm the identification of V2 as E. coli. This bacteria is sensitive to almost all the antibiotics tested, i.e. ampicilin, gentamycin, chloramphenicol, tetracycline, cephalothin, and clindamycin. Any of these antibiotics or a combination of these can be used to treat E. coli infections. E. coli is the most common bacteria of the human GIT, the first to inhabit it and also protect it (Hudault et al, 2001). Pathogenic strains of E. coli cause UTI, neonatal meningitis and gastroentitis (Levine1987). The identification procedure of bacteria isolated from faecal sample (F) began with it being gram negative which narrows down the search to gram negative bacteria. This also has a cell morphology of bacilli and therefore it can be categorised as a gram negative rod shaped bacteria. The colony morphology, round flat, smooth, small and yellow suggest it to be Salmonella. This is also supported by it being non haemolytic and non lactose fermenting, since F grows on MacConkey agar , but without colour change. Since it is catalase positive and oxidase negative it can be concluded that it belongs to the family of oxidase negative bacilli i.e. enterobacteriaceae. F grows on MacConkey agar, giving a positive result for lactose fermentation. This results helped in the conclusion that F can belong to either of the following genera: Edwardsiella, Erwinia, Morganella, Proteus, Providencia, Salmonella, Serratia, Shigella and Yersinia. Since F gave a negative test for urease and is also gamma hemolytic, it can most probably be Salmonella. F is sensitive to 5 of the 6 antobiotics tested: ampicilin, gentamycin, tetracyclin, chloramphenicol and cephalothin and any of these or a suitable combination can be used to treat Salmonella infection. This is also confirmed by the data available for antibiotic sensitivity of Salmonella. (Hirose et al, 2001). Salmonella is an enteric pathogen and is still a common cause of food borne infections (Tsai et al, 2007). It was first isolated by Theobald Smith in 1885 from pigs. The bacteria reaches man thrugh raw eggs, meat, vegetables and other food materia that may come in contact with faecal matter during production or transportation. It causes acute gastroentitis accompanied by nausea, vomiting and diarrhoea. The normal flora isolated from skin is a cocci with raised creamy colony, which can can most probably be S. epidermidis. S epidermidis is normal microflora of skin. It is non-pathogenic and causes infections only in case of major lapses in body defenses, such as intravenous drug use, catheter etc. The bacterial colonies obtained from the mouth are single cocci with round raised small colonies. This can either be S. mutans or S. salivarius. Both these are normal flora of the mouth. While S. mutans is present on the teeth, S. salivarius is present on inner cheek and gums. S. salivarius is one of the first bacteria to inhabit oral cavity after birth, while S. mutans comes after teething and remains till there are teeth. S. mutans is responsible for formation of plaque, production of acids and dental caries. Soap as well as handwash were found to be comparably effective agents, both giving zero growth on plate after washing of hands. However the results were not the same as class results, which showed the two cleaning agents to be comparably ineffective. The plate growth for class for both the agents was same before and after handwash. This could be due to differences in prewash condition of hands, washing techniques and plating techniques. Serial dilution of surface spray lead to turbidity which remained the same with consequtive dilutions, however the plate growth increased with each dilution reaching maximum with dilution 5. This was not the same as class results, in which turbidity of surface spray decreased with each dilution reaching zero for fifth dilution, and plate growth maximum for fourth dilution. Unlike surface spray, ethanol causes maximum disinfection in first dilution during which there is no plate growth, but with every consecutive dilution, turbidity as well as plate growth increases. Vinegar does not produce any turbidity with addition of water but shows an increasing plate growth with every dilution. Floor cleaner as well as bleach show minimum turbidity and plate growth in initial dilution, but both turbidity and plate growth increases with each dilution. All these agents are bactericidal, showing no growth at a specific dilution, minimum inhibition concentration (MIC) being different for different agents. For ethanol it is dilution 1, because ethnol works best in 70 to 90% aqueous solution. For the rest too, minimal growth is at dilution 1. Antibiotic resistance in various microorganisms is jeopardizing the health situation globally. Bacteria acquire resistance to various drugs by acquiring new genes that impart drug resistance to them by either of the following mechanisms: via horizontal gene transfer through: It is the major area of concern in development of multidrug resistance. It is the mechanism responsible for transfer of beta-lactamase gene in different bacteria. Many enterobacteria now carry this gene, thus making this class of antibiotics of no use in their control. The mechanisms of horizontal gene transfer are: 1 transformation, through plasmids (Mendez et al., 2009) : Exogenous genes are taken up throught he cell wall and incorporated as well as expressed as a part of the bacterial genome. 2 transduction (Blahova et al., 1999): Introduction of new genes in the bacteria via phage has been implicated in the transfer of antibiotic resistance to Pseudomonas aeruginosa. 3 transposons or jumping genes (Beringer et al., 1978): Transposons are genes capable of changing their locations within the genome. Tn 1403, a transposon from Pseudomonas strain was found to be made up three different transposon and thus illustrated the capability of transposons to impart multiple drug resistance in microbes (Stokes et al., 2004) By each of these processes, new genes are introduced into the microbe which impart resistance to the antibiotics eg. transposon Tn5 has been observed to impart drug resistance to Rhizobium (Stokes et al., 2004). by spontaneous mutations (Brodine et al., 1999): The evolutionary significance of mutations has been well documented. To microbes evolution is better adaptation to antimicobial agents, and several examples of such mutations are known, eg spontaneous resistance to fluoroquinolines is frequent in bacteria (Barnard and Maxwell, 2001) References 1 Barnard, F.M., and A. Maxwell. (2001). Interaction between DNA gyrase and quinolones: effects of alanine mutations at GryA subunit residues Ser83 and Asp87. Antimicrobial Agents and Chemotherapy, 45, 1994-2000. 2 Bergey, David H.; John G. Holt; Noel R. Krieg; Peter H.A. Sneath (1994). Bergeys Manual of Determinative Bacteriology (9th ed.). Lippincott Williams & Wilkins. 3 Beringer, J. E., Beynon, J. L., Buchanan-Wollaston & Johnston (1978). Transfer of drug resistance transposon Tn5 to Rhizobium. Nature, 276, 633-4. 4 Blahnova, J., Kralikova K., Kromery, V. & Bratonikova N. (1999). High frequency transduction of antibiotic resistance in Pseudomonas aeruginosa by a wild type Bacteriophage with restricted specificity for recipient strains. European journal of clinical microbiology and infectious diseases, 18(2), 152-4. 5 Brodine et al. (1999). Drug resistance patterns, genetic subtypes, clinical features, and risk factors in military personnel with HIV-1 seroconversion. Annals of Internal Medicine. 131, 502-6. 6 Chow, A. W., Gribble, M. J. & Bartlett, K. H. (1983). 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